Increasing ornithine accumulation to increase high mannose glycoform content of recombinant proteins

Abstract

The present invention relates to a method for manipulating the high mannose glycoform content of recombinant glycoproteins by regulating ornithine metabolism during cell culture.

What is claimed is: 
     
       1. A method of increasing high mannose glycoform content of a recombinant protein comprising culturing a host cell expressing the recombinant protein in a cell culture comprising ornithine or arginine, wherein ornithine production in the host cell is increased when compared to the host cell expressing the recombinant protein is cultured in a cell culture lacking ornithine or arginine, and
 the recombinant protein has an increased high mannose glycoform content than when the recombinant protein is expressed in the cell culture lacking ornithine or arginine. 
 
     
     
       2. The method of  claim 1 , wherein ornithine accumulation in the host cell is increased by the addition of at least 0.6 mM ornithine to the cell culture. 
     
     
       3. The method of  claim 1 , wherein the concentration of ornithine is selected from the group consisting of 0.6 to 14.8 mM; 6 to 14.8 mM; 0.6 mM, 6 mM, and 14.8 mM. 
     
     
       4. The method of  claim 1 , wherein ornithine accumulation in the host cell is regulated by the addition of at least 8.7 mM arginine to cell culture medium. 
     
     
       5. The method of  claim 1 , wherein the concentration of arginine is selected from the group consisting of 8.7 mM to 17.5 mM; 8.7 mM; and 17.5 mM. 
     
     
       6. The method of  claim 1 , wherein the host cell expressing the recombinant protein is cultured in a batch culture, fed-batch culture, perfusion culture, or combinations thereof. 
     
     
       7. The method of  claim 1 , wherein the host cell expressing the recombinant protein is cultured in a bioreactor. 
     
     
       8. The method of  claim 7 , wherein the bioreactor has a capacity selected from the group consisting of at least 500 L; 500 L to 2000 L; and 1000 L to 2000 L. 
     
     
       9. The method of  claim 1 , wherein the host cell expressing the recombinant protein is cultured in a serum-free cell culture medium. 
     
     
       10. The method of  claim 9 , wherein the serum-free culture medium is a perfusion cell culture medium. 
     
     
       11. The method of  claim 1 , wherein the host cells are mammalian cells. 
     
     
       12. The method of  claim 1 , wherein the host cells are Chinese Hamster Ovary (CHO) cells. 
     
     
       13. The method of  claim 1 , wherein the recombinant protein is a glycoprotein. 
     
     
       14. The method of  claim 1 , wherein the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine. 
     
     
       15. The method of  claim 1 , further comprising a step of harvesting the recombinant protein produced by the host cell. 
     
     
       16. The method of  claim 1 , wherein the recombinant protein produced by the host cell is purified and formulated into a pharmaceutically acceptable formulation.